Dynamin, a 100 kD GTPase, has been shown to be essential for receptor- mediated endocytosis. Cells over-expressing a dynamin mutant deficient in GTP binding accumulate clathrin coated pits at the plasma membrane, as do synaptosomes treated with GTP-gamma-S, and internalization of material by clathrin-coated vesicles is blocked under these conditions. A ring of electron-dense material localized at the necks of coated pits has been identified by immunoelectron microscopy as dynamin. Purified recombinant dynamin, under low salt conditions (<50mm), has been shown to form distinct stacks and rings with similar dimensions to the dense material seen in vivo at the necks of coated pits. This, together with previous work, suggests that dynamin molecules wrap around the constricting coated pit and upon GTP hydrolysis may undergo a concerted conformational change causing the release of coated vesicles into the cytosol. We are exploring the structure and function of dynamin by high resolution cryo-electron microscopy and biochemistry techniques. By lipid-layer crystallization techniques, we have shown that recombinant dynamin, purified from over-expression in baculovirus infected SF9 insect cells, binds to acidic phospholipids and forms helical tubes and two- dimensional arrays. We are currently optimizing conditions necessary for helical tube and 2-D crystal formation from which we plan to calculate 3-dimensional electron density maps of dynamin. In addition, we have also shown that, although GTP and GDP causes dynamin to disassemble, the addition of GDP + aluminum fluoride greatly enhances the assembly of dynamin into stacks as seen by negative- staining electron microscopy and as quantified by a sedimentation assay. This suggests that the transition state GDP + Pi locks dynamin into a rigid conformation which may play a crucial role in pinching off coated vesicles. Structural studies, including comparisons of 3 dimensional maps under different conditions (e.g. +/-GTP), and assembly assays will lead to further understanding of the mechanism of clathrin-coated vesicle budding.